Resistance to virus infection using modified viral movement protein

ABSTRACT

The present invention relates to the methods and nucleic acid compositions for the production of transgenic plants resistant to virus infection. In particular, it relates to transgenic plants comprising nucleotide sequences encoding dysfunctional viral movement protein (dMP) genes.

BACKGROUND OF THE INVENTION

The present invention relates to the methods and nucleic acidcompositions for the production of transgenic plants resistant to virusinfection. In particular, it relates to transgenic plants comprisingnucleotide sequences encoding dysfunctional viral movement protein (dMP)genes.

Local as well as systemic viral infection requires virus movement.Opportunistic introduction of viral particles occurs where cell wall andplasma membrane integrity has been disrupted, as for example throughmechanical damage caused by a biological vector such as an insect,nematode or fungus as-well-as abrasive forces such as the breaking of aleaf or branch. A progressive viral infection results if uponreplication the viral progeny is capable of spreading into adjacentcells and then systemically throughout the plant. In certain instancesinfectious virus have been shown as capable of replication in the hostcells but unable to move to adjacent healthy cells. When this occurs theinfection is said to be subliminal and the plant appears resistant.

The cell-to-cell spread of virus is not a passive process but requiresthe expression of a virus encoded product called a movement protein(MP). The movement proteins of many viruses have been tentativelyidentified as reviewed by Hull, R., Annu Rev Phytopathol 27: 213-2401989, and Maule, A. J., Crit Tev Plant Sci 9: 457-473 1991. The firstvirus-encoded movement protein (MP) identified was that of tobaccomosaic virus (TMV) (Deom et al., Science, 237 384-389 1987; and Meshi etal., EMBO J 6: 2557-2563 1987). Although dispensable for virusreplication, the 30 kDa MP of TMV is essential for cell-to-cell spreadof the infection (Deom et al., Cell 69: 221-224 1992). Furthermore,transgenic plants that express the TMV MP (MP(+) plants) can complementmutants of TMV that are movement deficient (Deom et al., supra 1987;Holt and Beachy, Virology 181: 109-117 1991). In both TMV-infectedplants and MP(+) plants the MP co-purifies with an insoluble cellularcomponent that contains cell walls (Deom et al., Proc Natl Acad Sci 87:3284-3288, 1990), and was localized by immunogold labeling to theplasmodesmata, the cytoplasmic connections between adjacent plant cells.Specifically, the TMV MP is localized to the central cavity of secondary(also referred to as modified primary) plasmodesmata which are formedthrough fusion of, and addition of, new protoplasmic bridges to primaryplasmodesmata (Ding et al., Plant Cell, 4: 915-928, 1992).

The mechanism(s) by which the MP potentiates virus movement from cell tocell is not fully understood. Wolf et al., Science 246: 377-379, 1989,demonstrated through dye coupling studies that the protein has a directeffect on the molecular size exclusion limit (SEL) of the plasmodesmata.Fluorescent dextrans with an average molecular mass of 9400 Da movedbetween cells of MP(+) transgenic plants, while the size exclusion limitof the MP(-) transgenic plants and non-transgenic plants was 700-800 Da.In a similar study a temperature-sensitive (ts) mutant of the MP wasunable to modify the SEL of plasmodesmata or to facilitate virusmovement at the non-permissive temperature (Wolf et al., Plant Cell 3:593-604, 1991).

Deletion mutants of TMV MP have been made and expressed in transgenicplants. Berna et al., Virology, 182: 682-689, 1991, studied transgenicplants that expressed truncated MP lacking up to 73 C-terminal aminoacids. The transformed plants were analyzed for MP subcellularlocalization and for complementation of the spread of a thermosensitiveTMV mutant Ls1. Ls1 is incapable of cell-to-cell movement at thenon-permissive (32° C.) temperature due to an inactivated MP. Deletionof the C-terminal 55 amino acids of MP had no effect on subcellularlocalization or complementation of Ls1. Deletion of an additional 19 aa(aa 195 to 213 aa) destroyed both cell localization and ability tocomplement Ls1.

Gafny, et al., Virology 187: 499-507 1992, studied a variety ofinfectious clones of TMV having N-terminal and C-terminal deletions inthe MP gene. The effect of the deletion mutations on local and systemicmovements of the infection was evaluated. Deletion of 9 to 33 C-terminalamino acids did not effect cell-to-cell movement as reflected by locallesion formation on Nicotiana tabacum cv. Xanthi NN plants. Deletion of55 C-terminal amino acids resulted in impaired movement and deletion of74 C-terminal amino acids resulted in a protein that could not supportvirus movement. In addition, MP deleted for N-terminal amino acids 3-5could not support virus movement.

Although a number of mutations in the TMV MP are found to alter theability of the protein to support virus spread, the effect suchmutations have on the production of a MP capable of blocking the spreadof virus infection was not determined. It is the determination thatdysfunctional MP can lead to viral resistance in plants, the resultantdysfunctional MP and the virus resistant transgenic plants that are thesubject of the present invention.

SUMMARY OF THE INVENTION

The present invention provides and claims methods for producing viralresistant transgenic plants that are transformed by a gene encoding amutated viral movement protein. The mutation causes the protein todysfunction as a virus movement protein but retain the ability tointeract with the plant constituents in such a manner as to block thespread of infectious virus.

The invention further provides the mutation is at the N-terminus of theprotein between amino acids 2 and 8 and preferably is a deletion ofamino acids 3, 4 and 5.

The dysfunctional virus movement protein is derived from a Tobamovirus,preferably tobacco mosaic virus, and is capable of inhibiting the spreadof viruses from a number of different groups or families including Ilarviruses which includes alfalfa mosaic virus, bromovirus, caulimorvirus,hordeivirus, luteovirus, tobamovirus, and tospovirus.

Plant species benefited by transformation with such a dysfunctionalvirus movement protein gene include, but are not restricted to tobacco,tomato, bean, potato, barley, wheat, cucumbers, melons and corn.

In general, the methods described herein are applicable to any virusmovement protein. Where the spread of a viral plant disease is theresult of a viral movement protein allowing for the intercellular and/orsystemic movement of a particular virus, this invention makes itpossible for the trained artisan to mutate and test the movement proteinand identify a dysfunctional movement protein that will interfere withvirus movement. In this regard, this invention further provides formutated viral movement proteins that interfere with virus movement.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the correlation of disease resistance with accumulation ofMPΔ3-5. (a) Xanthi nn plants were inoculated with TMV (0.25 μg/ml) andwere observed for systemic disease symptoms 9 DPI. Under the conditionsof these experiments symptoms were visualized on non-transgenic Xanthinn plants by 4-5 DPI. (b) Xanthi NN plants were inoculated with TMV (1.0μg/ml) and lesions were counted 48-72 h post-inoculation. Plants wereassayed for accumulation (Accum.) of MPΔ3-5 by a slot-blot immunologicalassay as described in the examples. Thirteen plants from each line arepresented; however, 45 plants from each line were tested in theexperiment. The MPΔ3-5 gene segregated 3:1 in both lines.

FIG. 2 shows the accumulation of MP and MPΔ3-5 in transgenic plants.Total cellular proteins were extracted from leaf tissues as described inthe examples. Leaves from three plants of each genotype were pooled foranalysis. The proteins were separated on a 12.5% polyacrylamide gel (50μg protein per lane), transferred to nitrocellulose and reacted withanti-MP antibodies followed by ¹²⁵ I-labeled secondary antibody. The MPand MPΔ3-5 were excised from the membrane and quantitated in a γcounter; c.p.m. are given below each lane. 2005 and 277 are transgenicplant lines that express the MP of TMV (Deom et al., supra 1987);3A5-NN-7B and 3A5-SX-11 are transgenic plant lines which express theMPΔ3-5; 3A7-SX-8 is a transgenic line that does not express the MPΔ3-5gene.

FIG. 3 shows the time course of development of systemic disease symptomsin plants inoculated with TMV or TMGMV. Seedlings of the Xanthi nntransgenic plant line 3A5-SX-11 that express the MPΔ3-5 gene 3A5-11(+)!,non-expressing transgenic plants 3A5-11(-)!, and non-transgenic Xanthinn plants (WT) were inoculated with: (a) 0.25 ug/ml TMV; (b) 1.0 ug/mlTMV; (c) 1.3 ug/ml TMV-RNA; (D) 2.0 ug/ml TMGMV. A plant was determinedto be systemically infected when it exhibited mosaic symptoms, or strongvein clearing, in more than one leaf.

FIG. 4 shows transgenic plants that accumulate the MPΔ3-5 develop lowernumbers, and smaller necrotic local lesions upon inoculation with TMVand TMGMV. Non-transgenic Xanthi NN plants and MPΔ3-5(+) plants (line3A5-NN-7B) were inoculated with (a) TMV (1.0 ug/ml) or (b) TMGMV (2.0ug/ml). Lesion sizes were measured at daily intervals. Between 60 and120 lesions were measured at each time point for the Xanthi NN plants,and 150-250 for the 3A5(+) plants. Bars represent standard error. HPI,hours post-inoculation.

FIG. 5 shows the number of leaves with symptoms of tobamovirus Obinfection as a function of time. One Leaf on each plant was inoculatedwith the tobamovirus! Ob with an IP that resulted in 14 lesions per leafon C. amaranticolor. NN (MP-)=◯ 2005 (MP+)=□, 3A5-NN-7B (dMP)=Δ,3A6-NN-2 (dMP)=⋄. *=At this day the symptoms for 3A5-NN-7B and 3A6-NN-2as compared to NN differ significantly at the 5% level.

FIG. 6 shows the number of leaves showing symptoms of TRV as a functionof time. One leaf on each plant was inoculated with TRV with an IP thatresulted in 260 lesions per leaf on C. amaranticolor. NN (MP-)=◯, 2005(MP+)=□, 3A5-NN-7B (dMP)=Δ, 3A6-NN-2 (dMP)=⋄. *=At this day the symptomsfor 3A5-NN-7B and 3A6-NN-2 as compared to NN differ significantly at the5% level.

FIG. 7 shows the number of leaves showing symptoms of AlMV as a functionof time. One leaf on each plant was inoculated with AlMV with an IP thatresulted in 25 lesions per leaf on C. amaranticolor. (a) NN (MP-)=◯,2005 (MP+)=□, 3A5-NN-7B (MPΔ3-5)=Δ, 3A6-NN-2 (MPΔ3-5)=⋄; (b) 3A7-SX-8(MP-)=, 274 (MP+)=▪, 3A5-SX-11 (MPΔ3-5)=▴. *=At this day the symptomsfor 3AS-NN-7B and 3A6-NN-2 as compared to NN or 3A5-SX-11 as compared to3A7-SX-8 differ significantly at the 5% level.

Definitions

"Amino terminus" refers to that end of the protein which has the freeprimary amine.

"Intercellular movement" refers to the passage of viral nucleoproteinand/or nucleic acid from cell to cell. Typically this passage occursthrough plasmodesmata which are modified by viral movement proteins topermit viral transfers.

"Movement protein" are viral proteins which bind to the plant cell wallsand permit or facilitate travel of infectious viral components from cellto cell through the plasmodesmata. They are sometimes referred to aswild type (wt) movement proteins or MP when the text attempts todifferentiate functional movement proteins from mutated movementproteins.

"Mutated movement proteins" are movement proteins that have beenmodified to inhibit the travel or passage of viral components throughthe plasmodesmata. They are also referred to as dysfunctional movementproteins or dMP. **

"Bind the cell walls" refers to the interaction that is predominantlymore with the cell wall, plasma membrane or plasamadesmata of a plantrelative to the other cellular components. Typically, with any commonlypracticed method of plant cell fractionation that results in a cell wallenriched fraction, a larger percentage of the cellularly expressedmutated movement protein will be associated with the cell wall fraction.The cell wall fraction will contain fragments of plasma membrane andplasmodesmata. The mutated movement protein can also be defined ashaving subcellular localization to the cell wall, plasma membrane orplasmodesmata or accumulated at the cell wall, plasma membrane orplasmodesmata.

"Viral resistant plant" refers to a plant that resists the cell-to-cellspread of virus particles. The cell-to-cell spread of virus infectioncan be systemic where by symptoms of infection develop at a site on theplant distant from the initial inoculation site, or virus spread may berestricted to an area of necrosis where by symptoms of infection areexemplified by local lesions. In addition, the cell-to-cell spread ofvirus can remain localized near the inoculation site without thedevelopment of lesions. In an assay, resistance that is more than 40% ofthe resistance of the control in either by symptom intensity or viraltiter measured by immunoassay, direct examination under an electronmicroscope, qualitative assay such as a bioassay on a reporter plant(e.g. C. amaranticolor) or by other quantification means.

DETAILED DESCRIPTION

This invention provides transgenic plants resistant to viral infection.Virus resistance results from the transgenic expression of a geneencoding a dysfunctional virus movement protein. The dysfunctionalmovement protein confers resistance to the plant by competing with andblocking the wild type movement protein of the otherwise infectingvirus. The invention describes methods for mutating viral movementprotein genes, transforming plants with the mutated gene and analyzingthe transgenic plants for resistance to virus infection and spread. Inaddition, transient assays are provided that allow for the rapidselection of promising dysfunctional movement proteins that can betested further in transgenic plants.

The mutation of wild type movement proteins to produce novel mutantmovement proteins is done by conventional means. Movement proteins areidentified genetically by analyzing the viral genome for gene sequences,which when altered, restrict the ability of the virus to spread locallyor systemically, but do not alter the ability of the virus to replicateor assemble. A number of movement proteins have been described in theliterature. They include those of tobacco mosaic tobamovirus (Deom etal, 1987, supra); cowpea mosaic comovirus (van Lent, Wellink, and vanKammen, J Gen Virol 71: 219, 1989); brome mosaic bromovirus (Sacher andAhlquist, J Virology, 63: 4545, 1989); red clover mottle virus (Shankset al, Virology, 173: 400, 1989).

The exemplified mutant movement protein disclosed herein is a 3-5 aminoacid deletion (MPΔ3-5). Other mutations are possible based upon theteachings herein. It is not possible to describe all the possiblemutations from each viral movement protein that will interfere withviral passage from cell to cell. One of skill could readily providenumerous conserved substitutions in the exemplified mutated movementprotein. For example the neutral amino acids could be substituted withglycine or alanine and aspartic acid residues could be changed toglutamic acid. There is no limit to the number of conservedsubstitutions or additions or deletions that could be tested and asubstantial number of such modified proteins would be the equivalent ofthe mutated (dysfunctional) TMV movement protein described herein. Aminoterminal deletions are preferred however amino acid substitution wouldalso function to provide suitable mutations.

There are various means for producing mutations. Routine recombinantmethods are the most convenient means. Single site mutations can beintroduced by a variety of routine methods such as the M13 system. Mostpreferably, fragments of synthetic nucleic acid containing the desiredmutations can be introduced by a combination of endonuclease cleavageand ligation. Controlled nuclease activity can be used to deleteportions of the wild type gene (Maniatis et al., Molecular Cloning: Alaboratory manual; Cold Spring Harbor Laboratory, Press, Cold SpringHarbor, N.Y.). Alternatively, point mutations and small deletions aremade by directed mutagenesis reactions (Nakamaye and Eckstein, NucleicAcids Research, 14: 9679-9698, 1986).

The mutations must retain the ability of the movement protein to bind tothe plasma membrane and the plant cell wall while producing the desiredinhibitory effect of blocking the wild type movement protein frompermitting passage of infectious viral components from cell to cell. Inaddition, such mutations must not disrupt the intracellular stability ofthe protein and make it susceptible to the degradative pathways of theplant cell. It is known that the TMV movement protein is sensitive tosubstantial mutations. If you delete amino acids 3-8 of the protein, thestructure of the molecule is sufficiently altered that the ability ofthe protein to bind cell walls is effected and resulting protein whiledysfunctional and mutated may not be useful as a viral inhibitory agent.

For this reason, once the mutated proteins are produced each proteinmust be tested in accordance with the methods and assays producedherein. Following the teachings presented herein a significant number ofmutated movement protein can be obtained that are suitable forinhibiting the cell to cell spread of viral infection. Suitablemutations for use in this invention are readily identified by routinetitration experiments. For example, Table 1 lists a number of threeamino acid deletions (TAD) of the movement protein that were made inTMV. The infectious potential of the mutated virus was studied ontransgenic (MP+) and wild type systemic and local lesion host, Xanthi NNand Xanthi nn, respectively, of Nicotiana tabacum. Proteins 1-4 and 9are examples fitting the criteria for conferring virus resistance.

Subcellular localization was determined with a transgenic plantexpressing a functional movement protein deleted of 26 amino acids atthe C-terminus, (Berna et al., 1991, supra). The viral expressed mutatedmovement protein was detected in cell wall enriched fractions usingSDS-PAGE electrophoresis and western immunoblot analysis, the viralmovement protein discerned from the transgenically expressed movementprotein on the basis of size. Those virus that were infectious onlyafter complementation by the MP of the transgenic plant and showedsubcellular localization to the cell wall were considered to have dMPthat would provide resistance when transformed and tested in transgenicplants. This experimental strategy and modifications thereof that arereadily apparent to those of skill can be employed to identify othermutations of any plant viral movement protein for use in this invention.

In this regard, this invention encompasses virus that encode mutatedmovement proteins where the virus are infectious after complementationby a functional movement protein, and preferably are found to express amutated movement protein that binds preferentially to the cell wall. Thecomplementing functional movement protein does not necessarily need tobe wild type movement protein but can be any movement protein altered byany means either genetically or chemically while retaining nativefunction.

                                      TABLE 1    __________________________________________________________________________    Function of three AA deletions (TAD) of the MP in tobacco mosaic    virus infections.                    Genotype of the host:*              Position of                    Xanthi NN                          Xanthi nn                                  Subcellular    TAD       Deleted AA              Deleted AA                    MP+                       WT MP+                             WT   localization    __________________________________________________________________________     1 Val, Asn, Ile               9-11 +  -  +  -    CW     2 Lys, Met, Glu              19-21 +  -  +  -    CW     3 Thr, Pro, Val              29-31 +  -  +  -    CW     4 Val, Asp, Lys              39-41 +  -  +  -    CW     5 Ser, Leu, Ser              49-51 +  -  +  -     6 Val, Lys, Leu              59-61 +  -  +  -     7 Leu, Ala, Glu              69-71 +  -  +  -     8 Asn, Leu, Pro              79-81 +  -  +  -     9 Val, Ser, Val              88-90 +  -  +  -    CW    10 Arg, Ala, Asp               99-101                    +  -  +  -    11 Tyr, Thr, Ala              109-111                    +  -  +  -    12 Phe, Lys, Val              119-121                    +  -  +  -    13 Thr, Gln, Asp              129-131                    +  -  +  -    14 Val, Leu, Val              139-141                    +  -  +  -    15 Ser, Ala, Gly              149-151                    +  -    16 Phe, Val, Ser              159-161                    +  -    17 Asn, Ile, Lys              169-171    18 Thr, Asn, Val              179-181    19 Leu, Thr, Glu              189-191                    +  +  +  +    20 Glu, Asp, Val              199-201    21 Lys, Phe, Arg              209-211                    +  -  +  -    22 Asp, Val, Arg              219-221                    +  +    23 Asp, Arg, Ser              229-231                    +  -  +  -    24 Asn, Val, Lys              239-241                    +  +  +  +    ND    25 Lys, Lys, Asn              249-251                    +  +  +  (m)+ .sup.                                  ND    26 Glu, Ala, Thr              259-261                    +  +  -  -    ND    __________________________________________________________________________     * `+` = infection     `-` = no infection     TAD = Three amino acid deletion;     AA = Amino acids;     WT = wild type.     Xanthi NN expressing the MP gene (MP+) = line 2005.     Xanthi expressing the MP gene (MP+) = line 277.     m = mild     ND = Not done.

In addition, there are described herein transient assays that detectcompetition between wild type and dysfunctional movement protein thatare simultaneously expressed in a single plant either from a singleviron or infectious transcript containing a simultaneous expressioncassette or as separate co-inoculated infectious clones. If the mutatedmovement protein functions to inhibit the movement of virus, it can bepresumed that the mutated movement protein is a part of this invention.

A. Methods of Virus Propagation

In general, the invention is applicable to any species of virus thatrequires a movement protein to spread infection either systemically orlocally. Plant virology is a well developed area of technology. Thereare routine methods for propagating plant viruses as described in(Matthews, ed. "Diagnosis of Plant Virus Diseases," CRC Press, BocaRaton, Fla., pp 130-152, 1993). Virus identification is done byinoculation to diagnostic host plants and by reactions to virus specificantibodies, methods commonly used in the field of plant virology. Manycommonly identified plant virus are available from the American TypeCulture Collection.

B. Methods of Genetic Analysis and Manipulation

Generally, the nomenclature and the laboratory procedures in recombinantDNA technology described below are those well known and commonlyemployed in the art. Standard techniques are used for cloning, DNA andRNA isolation, amplification and purification. Generally enzymaticreactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like are performed according to the manufacturer'sspecifications. These techniques and various other techniques aregenerally performed according to Sambrook et al., Molecular Cloning--ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989.

The identification and isolation of a viral movement protein gene from aparticular virus may be accomplished by a number of techniques. Assummarized in Table 1 from Hull, supra, 1989, there is evidence that alarge number of virus groups including, alfalfa mosaic, bromovirus,caulimovirus, comovirus, cucumovirus, dianthovirus, geminivirus,hordeivirus, ilarvirus, nepovirus, potexvirus, potyvirus, tobamovirus,tobravirus and tospovirus have viral-coded cell-to-cell movementproteins. Such evidence is the result of experiments involvingsubliminal infection, complementation, mutation, cytology and sequencehomology.

In general, using techniques designed to clone virus genomes (see, e.g.,Sambrook et al., 1989, supra) one can obtain a cDNA clone of aparticular virus, and through sequence analysis define putative openreading frames which can be subcloned for further studies. Antibodiesraised to oligopeptides of the predicted protein sequences can then beused to analyze for protein expression during the virus infection cycle,to determine protein interaction with different cellular componentsusing cell fractionation studies, and to directly observe and localizethe movement protein through electron microscopy and immunogold labelingof thin sections of infected plant tissues. Detailed descriptions ofsuch techniques as well as further reference to experimental materialsand methods can be found in Hull, supra, 1989, incorporated herein byreference.

C. Methods of Transferring Genes to Plants

a. Agrobacterium-Mediated Transfer

This approach provides a routine and efficient system to transfer dMP toa variety of plant species. Agrobacterium tumefaciens-meditatedtransformation techniques are the most commonly used techniques fortransferring genes into plants.

All species which are a natural plant host for Agrobacterium aretransformable in vitro. Most dicotyledonous species can be transformedby Agrobacterium. Monocotyledonous plants, and in particular, cereals,are not natural hosts to Agrobacterium. There is growing evidence nowthat certain monocots can be transformed by Agrobacterium. Using novelexperimental approaches cereal species such as rye (de la Pena et al.,Nature 325: 274-276, 1987), corn (Rhodes et al., Science 240: 204-207,1988), and rice (Shimamoto et al., Nature 338: 274-276, 1989) may now betransformed.

The transfer of genetic material is brought about by a segment of the Tiplasmid carried by A. tumefaciens known as T-DNA. When the T-DNA istransferred to the nuclear genome of a susceptible plant transformationoccurs. As it naturally occurs on the Ti plasmid, the T-DNA is flankedby 25 bp direct repeats and contains genes that encode enzymes involvedin the synthesis of auxin and cytokinin, the over production of whichleads to uncontrolled cell proliferation and resultant transformation.By disarming the auxin and cytokinin genes and inserting a gene ofinterest, the T-DNA becomes a vehicle by which genetic sequences can bestably transferred into plants.

Regions of the T-DNA have been cloned in E. coli and used to constructplasmids that carry markers for selection and propagation in bothbacteria and transgenic plants and are capable of homologousrecombination with disarmed resident Ti plasmids in A. tumefaciens. Suchplasmids are designed to carry transcriptional promoters that will drivethe expression of a selected gene in the transformed plant. For example,pMON316 is a plasmid that contains drug resistance markers for selectionand cloning in E. coli, i.e., spectinomycin/streptomycin (Spc/Str); amarker for selection in transformed plants, i.e., neomycinphosphotansferase type II (Npt II), and a nopaline synthase gene (NopSyn); a region of homology for recombination with a resident Ti plasmidin A. tumefaciens; and the CaMV 35S promoter and NOS 3' untranslatedregion separated with a polylinker into which a gene of interest can beinserted, (Sanders et al., Nucl Acids Res, 160: 363-371, 1987).

Detailed procedures for Agrobacterium-mediated transformation ofdifferent plant species have been described by Horsch, et al., Science,227: 1229-1231, 1985; Ulian, et al., In Vitro Cellul Dev Biol 24:951-954, 1988; McGranahan, et al., Bio/Technology 6: 800-804, 1988;Fillati, et al., Populus, Mol Gen Genet 206: 192-199, 1987; James, etal., in Genetic Engineering of Crop Plants, 49th Nottingham EasterSchool (G. Lycett and D. Grierson, eds.), pp. 239-248, Sutton Bonington,University of Nottingham, Butterworths, UK, 1990.

b. Electroporation

In this technique, protoplasts are made by removing the plant cell wallusing hydrolytic enzymes in an osmotically balanced solution. Bysubjecting the protoplasts, while in a solution of DNA, to a sharpdischarge of electricity, pores open on the plasmalemma of theprotoplast through which the DNA can enter. A small fraction of the DNAintegrates into the plant's chromosomes and result in protoplasts thatstably express the gene through which transformatin is confirmed. Oncestable transformation is established, the protoplasts are treated topromote cell wall regeneration and further development to a whole plantwhich will then yield transgenic plants. General techniques for makingand electroporating protoplasts from woody plants are published byRevilla et al., Plant Sci 50: 133-137, 1987.

c. Biolistic Method

This technique involves high velocity penetration of the plant cell wallwith dense particles of gold or tungsten coated with DNA. Detaileddescriptions of this technique is published by Klein et al., Nature 327:70-73, 1987.

D. Methods of Transgenic Plant Selection and Propagation

After transformation, transformed plant cells or plants comprising theintroduced DNA must be identified. A selectable marker, such as thosediscussed above is typically used. Transformed plant cells can beselected by growing the cells on growth medium containing theappropriate antibiotic. The presence of opines can also be used if theplants are transformed with Agrobacterium.

After selecting the transformed cells, one can confirm expression of thedesired heterologous structural gene using a number of differenttechniques. Simple detection of mRNA encoded by the inserted DNA can beachieved by well known methods in the art, such as Northern blothybridization. One can us immunoprecipitation or Western blot analysisif antibodies to the desired protein are available. The insertedsequence can be identified using the polymerase chain reaction (PCR) andSouthern blot hybridization, as well. Detailed description of thesetechniques are found in Sambrook, 1989, supra.

Transformed plant cells (e.g., protoplasts) which are derived by any ofthe above transformation techniques can be cultured to regenerate awhole plant which possesses the transformed genotype and thus thedesired viral resistant phenotype. Such regeneration techniques rely onmanipulation of certain phytohormones in a tissue culture growth medium.Plant regeneration from cultured protoplasts is described in Evans etal., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,pp. 124-176, MacMillilan Publishing Company, New York, 1983; andBinding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRCPress, Boca Raton, 1985. Regeneration can also be obtained from plantcallus, explants, organs, or parts thereof. Such regeneration techniquesare described generally in Klee et al. Ann. Rev. of Plant Phys.38:467-486, 1987.

Accumulation of dMP in the leaves of transgenic plants indicates stableexpression and possible resistance. As a preliminary analysis for theexpression of dMP in transgenic plants rapid screening methods thatidentify plants that accumulate MP can be developed. For example, leaftissue can be ground and soluble proteins extracted by standard means.The proteins can be absorbed to nitrocellulose and detection made usinglabeled antibodies specific for the protein of interest.

E. Methods for Testing Virus Resistance

To test for resistance to virus infection the transgenic plant isinoculated with a requisite virus and observed overtime on leaves thedevelopment of disease symptoms. For example, TMV resistance istypically tested on cultivars of Nicotiana tabacum. In particular,systemic spread of infection maybe evalualted using a systemic host suchas cultivar Xanthi nn, and infection that is restricted to an area ofnecrosis maybe evaluated using a local lesion host such as cultivarXanthi NN. Systemic hosts, i.e. Nicotiana tabacum cvs. Xanthi nn is asystemic host for TMV, and hosts in which infection is restricted to anarea of necrosis, i.e. cvs. Xanthi NN, a "local lesion" host for TMV,can be used to test for virus resistance. With systemic hosts inoculumis applied to a select leaf, preferably a lower mature leaf, anddevelopment of disease symptoms is observed for over time on leavesdistant from the inoculation site, such as the developing younger upperleaves. With a local lesion host, the number of necrotic local lesionsare counted per inoculated leaf. Inoculation is done by disrupting theprotective outer layers of plant tissue, as for example by rubbingleaves with carborundum, and applying the virus. In addition to usingwhole virus, the viral RNA can also act as the infectious agent.

The following examples are provided by way of illustration only and notby way of limitation. Those of skill will readily recognize a variety ofnoncritical parameters which could be changed or modified to yieldessentially similar results.

EXAMPLES Example 1 Preparation and Analysis of Transgenic Plants thatExpress Dysfunctional Viral Movement Proteins and are Resistant to theSpread of Virus Infection

This example describes how to produce transgenic plants resistant to thespread of virus infection. Nicotiana tabacum cvs. Xanthi nn and XanthiNN plants were transformed with a chimeric gene encoding a dysfunctionaltobacco mosaic virus movement protein that lacks amino acids 3, 4 and 5(MPΔ3-5). Tissue fractionation studies showed only a low level of MPΔ3-5accumulated in the cell wall-enriched fraction compared with transgenicplants that accumulate wild-type MP. In addition, dye coupling studiesshowed that MPΔ3-5 enabled the movement between leaf mesophyll cells ofa fluorescently labeled dextran of 3 kDa, while 9.4 Kda molecules failedto move. This is in contrast to transgenic plants expressing thewild-type MP gene where the 9.4 kDa probe did move from cell to cell.Viral resistance was studied by inoculating the seedings fromself-fertilized transgenic plants with either; TMV, TMV-RNA, or thetobamoviruses TMGMV (tobacco green mosaic virus) or SHMV (sunnhempmosaic virus), and observing the development of disease symptoms. Insummary, in all instances disease development was inhibited intransgenic Xanthi NN and Xanthi nn plants expressing the MPΔ3-5 gene.

a) Genetic Manipulations and Preparation of Dysfunctional Viral MovementProtein

DNA manipulations such as plasmid preparation, restriction, and ligationwere essentially as described by Sambrook et al., 1989, supra. DNAsequencing was done as described by Tabor and Richardson, Proc Natl AcadSci USA 84, 4767-4771, 1987. Transcription and translation reactionswere performed using T7 RNA polymerase and wheat germ extract purchasedfrom Promega Corporation, Madison, Wis., according to the manufacturer'sinstructions. RNA was extracted from plants as described by Logemann etal., Anal. Biochem. 163, 16-20, 1987.

Site-directed mutagenesis of the cloned cDNA encoding the TMV MP isdescribed in Gafny, R., et al., 1992, supra. Briefly, N-terminaldeletions as that found in the MPΔ3-5 gene were constructed using the invitro mutagenesis system purchased from Amersham Corporation, ArlingtonHeights, Ill. The MP gene originated from plasmid pTM934 described byOliver, M. J., et al., Virology 155, 277-283, 1986. The EcoR1-BamH1fragment from PTM934 was cloned into the EcoR1-BamH1 sites ofpBluescript KS+ purchased from Stratagene, La Jolla, Calif. Singlestranded DNA was prepared and used as a template for site-directedmutagenesis. Synthetic oligonucleotides corresponding to 16-20nucleotides from the 5' and the 3' boundaries of the deletion were usedto mutate the MP gene. The amino-terminal first two amino acids of theMP were retained to preclude impairment in translation initiation.

The cloned cDNA encoding the MPΔ3-5 protein was ligated into theintermediate plasmid pMON316 described by Sanders, P. R., et al., 1987,supra, between the CaMV 35S promoter and the nopaline synthase 3'untranslated region. Agrobacterium tumefaciens-mediated gene transferwas done as describe by Horsch, R. B., et al., 1985, supra, was used tointroduce the MPΔ3-5 gene into N. tabacum cvs. Xanthi nn and Xanthi NNas previously described by Deom, C. M., et al., 1990, supra.

b) Characterization of Transgenic Plants Expressing the DysfunctionalViral Movement Protein Gene

Two tobacco cultivars were used for transformation experiments with theMPΔ3-5 gene, N. tabacum cvs. Xanthi nn, a systemic host for TMV, andXanthi NN, a host in which infection is restricted to an area ofnecrosis, i.e., a "local lesion" host. Eleven parental (R_(O)) Xanthi NNand 20 Xanthi nn plants were regenerated and taken to seed (R₁ seeds).Plants were grown in a growth room under artificial light (14 h light/10h dark period) at 25-30° C.

Plants were analyzed for accumulation of MPΔ3-5 protein using thefollowing procedure with the results shown in FIG. 1. Twenty milligramsof leaf tissue was ground in 100 μl of extraction buffer (75 mMTris-Hcl, Ph 6.8, 6 M guanidine-Hcl), incubated in boiling water for 5min, and the insoluble material was discarded following centrifugationfor 5 min in a microcentrifuge. The soluble extract was applied tonitrocellulose with a slot-blot apparatus, reacted with anti-MPantibodies, Deom et al., 1987, supra, and then with ¹²⁵ I-labeled donkeyanti-rabbit serum purchased from Amersham, supra. For experiments thatrequired greater amounts of material, total cellular proteins wereextracted by grinding 1 g of leaf tissue in 2 ml extraction buffer,incubated in boiling water for 5 min, and the insoluble material wasdiscarded following centrifugation for 10 min at 10,000 g.

To determine if the MPΔ3-5 protein expressed by the transgenic plantswere capable of inferring viral resistance, five plants from each linewere inoculated with TMV and observed for delay or inhibition of localor systemic disease symptom development with the results shown in table2.

Where inoculation is referred to here as well as throughout theexamples, the leaves were mechanically inoculated using carborundum asan abrasive. The inoculum, i.e., TMV, TMV-RNA, TMGMV, or SHMV, wasdiluted to the appropriate concentration in inoculation buffer (20 Mmphosphate, Ph 7.2, 1 mM EDTA). After inoculation with 100 μl per leafthe leaves were rinsed with water and the plants were observed daily fordisease symptoms.

Progeny of six of the 12 transgenic Xanthi NN plant lines and 16 of the20 transgenic Xanthi nn plant lines, exhibited a degree of inhibition ofviral spread based upon the criteria shown. Furthermore, it was foundthat reduction of spread of infection correlated with accumulation ofMPΔ3-5 protein as shown in FIG. 1 and table 2.

R1 progeny of two transgenic tobacco lines, 3A5-NN-7B (Xanthi NN line)and 3A5-SX-11 (Xanthi nn line) were chosen for further analysis. Theaccumulation of MPΔ3-5 segregated in progeny of 3A5-SX-11 with a 3:1ratio (MPΔ3-5(+):MPΔ3-5(-)!, suggesting that the MPΔ3-5 gene isexpressed from a single locus in this plant line. The segregation ratioin seedlings from line 3A5-NN-7B was 59:1, suggesting that the MPΔ3-5gene is expressed from three loci.

To test if the MPΔ3-5 protein was capable of supporting the spread ofvirus, plants that accumulated the MPΔ3-5 were inoculated withtranscripts of a cDNA clone of TMV that does not express the MP due to aframe-shift mutation in the MP gene. The cDNA clone is described by Holtand Beachy, 1991, supra. As expected, these plants did not complementthe spread of the movement-deficient TMV mutant and no disease symptomswere produced.

                  TABLE 2    ______________________________________    Analysis of MPΔ3-5 transgenic plant lines    (a) Xanthi nn plants                 Number of plants (out of five)                   Accumulating                              Showing    Plant line     MPΔ3-5                              resistance    ______________________________________    3A5-SX-2       4          4    3A5-SX-3       5          4    3A5 -SX-4      5          5    3A5-SX-5       0          0    3A5-SX-8       5          5    3A5-SX-10      3          2    3A5-SX-11      5          4    A5-SX-12       5          5    3A6-SX-1       4          4    3A6-SX-5       4          3    3A6-SX-6       2          2    3A6-SX-7       5          5    3A7-SX-2       4          4    3A7-SX-4       4          4    3A7-SX-5       4          4    3A7-SX-6       2          2    3A7-SX-7       1          0    3A7-SX-8       0          0    3A7-SX-9       3          3    3A7-SX-9A      0          0    Xanthi nn      /          0    ______________________________________    (b) Xanthi NN plants                    No. of  Percentage of    Plant line      lesions control    ______________________________________    3A5-NN-2        17 (2)  22.7    3A5-NN-4        39 (41) 52    3A5-NN-7B       19 (16) 25.3    3A5-NN-10A      56 (28) 74.7    3A5-NN-10B      12 (6)  16    3A6-NN-2         7 (2)  9.3    3A6-NN-9        24 (5)  32    3A6-NN-10       54 (37) 72    3A6-NN-11       41 (4)  54.7    3A7-NN-1        35 (7)  46.7    3A7-NN-4        17 (2)  22.3    3A5-NN-2        85 (21) 113.3    Xanthi NN       75 (37) 100    ______________________________________     (a) Five plants from each transgenic line were inoculated with TMV (0.5     μg/ml). The values given are the number of plants out of the five     tested that accumulate the MPΔ35, and that show resistance.     Accumulation was determined by slotblot analysis of each plant as     described in the examples. Plants that did not express systemic symptoms     by 8 DPI were considered to be resistant. Control plants showed disease     symptoms by 4-5 DPI.     (b) Ten plants from each transgenic Xanthi NN line were analyzed for     resistance. The values given are the average number of necrotic local     lesions per inoculated leaf counted 48-72 h after inoculation of plants     with TMV (1.0 μg/ml). Values in parentheses represent the standard     error. Percentage of control was calculated as the percentage of the     number of lesions counted on the control Xanthi NN plants.

c) Determination of the Subcellular Accumulation of DysfunctionalMovement Protein

The accumulation of the MPΔ3-5 gene product expressed in transgenicplant tissue was analyzed following subcellular fractionation andWestern immunoblot analysis of proteins in each fraction and comparedwith similar studies using plant line 277, a transgenic plant previouslyshown to accumulate wild-type MP, (Deom et al., 1990, supra).

Subcellular fractionation was done as described by Deom, et al., 1990,supra. As shown in Table 3, approximately 90% of the WT MP co-purifieswith an insoluble cellular component that largely contains cell walls(CW), the remaining 10% is in the membrane-rich fraction (P30) or in thesoluble supernatant (S30). In contrast, the MPΔ3-5 protein wasessentially evenly distributed between the cell wall and P30 fractions.

                  TABLE 3    ______________________________________    Relative levels of wild-type MP and MPΔ3-5 in subcellular    fractions from leaf tissue    Relative levels of MPΔ3-5, c.p.m. × 10.sup.-3 g FW.sup.-1    Plant line            CW            P30        S30    ______________________________________    277.sup.a            1635 (91.4)   147 (8.2)  5 (0.3)    3A5-SX-11            83 (41.8)     103 (52)   12 (6.2)    3A5-NN-7B            75 (38.3)     113 (57.8) 7 (3.9)    ______________________________________     The values are given in c.p.m. × 10.sup.-3 of .sup.125 Ilabeled     secondary antibody bound to immunoblots and represent the relative amount     of MPΔ35 or MP per gram fresh weight (FW) of leaf tissue. Leaves     from three plants of each genotype were pooled for analysis. Values in     parentheses represent the percentage of the total MP that is represented     in the subcellular fraction. Proteins were extracted from fractionated     leaf tissue,  #separated on a 12.5% polyacrylamide gel, transferred to     nitrocellulose and reacted with antiMP antibodies followed by .sup.125     Ilabeled secondary antibody, as described in the examples. C.P.M. were     determined after excising and counting each sample in a τ counter. CW     cellwall-enriched fraction (20 μg protein analyzed per lane); P30,     membranerich fraction (100 μg protein analyzed per lane); S30, soluble     fraction (200 μg protein analyzed per lane).     .sup.a Plant line 277 expresses a wildtype TMV MP.

To compare the amounts of NP and MPΔ3-5 that accumulated in transgenicplants, total cellular proteins were extracted from leaf tissue bygrinding two 5.9 mm diameter leaf disks per 50 μl grinding buffer (35 Mmpotassium phosphate, pH 7.5, 10 Mm β-mercaptoethanol, 400 Mm NACl) andthe insoluble material was discarded by centrifugation for 10 min in amicrofuge. Samples of leaves from three plants of each genotype werepooled for analysis. The proteins were separated on a 12.5%polyacrylamide gel (50 ug protein per lane), transferred tonitrocellulose and reacted with anti-MP antibodies followed by ¹²⁵ Isecondary antibody. The anti-MP antibodies were prepared as described inDeom, et al., 1987, supra, and the ¹²⁵ I-labeled donkey anti-rabbitserum was purchased from Amersham. The bands corresponding to MP andMPΔ3-5 were excised from the membrane and quantitated in a τ counter;c.p.m. are given below each lane. In addition to the transgenic plantline describe above, plant line 2005 is a Xanthi NN line that expressesthe WT MP gene described in Deom et al., 1987, supra; 3A5-NN-7B and3A5-SX-11 are transgenic plant lines which express the MPΔ3-5; 3A7-SX-8is a transgenic line that does not express the MPΔ3-5 gene. As shown inFIG. 2, in the two transgenic plant lines tested, MPΔ3-5 accumulated tobetween 25 and 40% of the level of accumulation of WT MP in transgeniclines 277 and 2005.

d) Differences Between Transgenically Expressed WT-MP and DysfunctionalMovement Protein with Respect to Moleculer Size Exclusion Limits (SEL)of the Plasmodesmata for Sized Fluorescently Labeled Dextrans

For microinjection studies, plants were grown in a controlledenvironment growth chamber for 3 to 4 weeks post-transplantation beforebeing used. The temperature regime in the growth chamber was 24° C./18°C. (day/night) with a 16 h photoperiod at a PAR level of 230-280 μmolM⁻² sec⁻².

The plasmodesmal SEL in various tobacco lines was determined bymonitoring intercellular coupling of microinjected fluorescent probes.The probes used were lucifer yellow CH (LYCH) with a molecular mass of457 Da and fluorescein isothiocyanate-conjugated dextrans (F-dextrans)with molecular masses ranging from 3 to 10 kDa. These probes aredescribe in detail by Wolf S., et al., 1989, supra. All probes weredissolved in 5 Mm KHCO₃ and stored at 4° C. The experimental system andprocedures used for microinjection have been previously described inDing et al., 1992, supra. Dye movement was monitored using anepifluorescence microscope (Leitz Orthoplan, Ernst Leitz GMBH, Wetzler,Germany) equipped with a blue (BP 390-490) excitation filter, achlorophyll cutoff filter, and a video-intensified microscopy system(model C1966-20, Hamamatsu Photonics K.K., Hamamatsu City, Japan).

As shown in Table 4, in MP(+) plants the 10 kDa F-dextran readily movedout of injected cells into neighboring cells. However, in MPΔ3-5(+)plants, while the 3 kDa F-dextran moved from cell to cell, the 10 kDaF-dextrans failed to move. In MPΔ3-5(-) transgenic plants, neither the 3kDa nor the 10 kDa F-dextran moved out of the injected cell. However,lucifer yellow CH (LYCH), with a molecular mass of 457 Da, moved freelyfrom cell to cell in all the plant lines, regardless of MP geneexpression (Table 4).

                  TABLE 4    ______________________________________    Mobility of fluorescent probes between leaf mesophyll cells of    various tobacco lines                        F-dextran    Plant line.sup.a               Leaf age.sup.b                        LYCH      3 kDa 10 kDa    ______________________________________    277         6th     --        --    5 (5)    3A5-SX-11  5-7th    5 (5)     4 (5) 0 (5)    3A7-SX-8   5-6th    5 (5)     0 (5) 0 (5)    WT         10th     5 (5)     0 (5) 0 (5)    ______________________________________     The data are presented as number of experiments manifesting cellto-cell     dye movement versus total number of experiments (in Parentheses). In each     experiment two leaves from each plant were injected at two to three     injection sites per leaf as a minimum. The different experiments were     performed at different dates with different plants.     .sup.a 277 expressed the wildtype MP of TMV; 3A5SX-11 expresses     MPΔ35; 3A7SX-8 was transformed with, but did not express, the     MPΔ35 gene; WT was nontransgenic Xanthi nn tobacco plants.     .sup.b The first leaf was defined as the youngest to attain a length of 5     cm.

e) Analysis of the Spread of Virus in Transgenic Plants that AccumulateDysfunctional Viral Movement Protein

For these studies, TMV (tobacco mosaic virus, U1 strain) and TMGMV(tobacco mild green mosaic virus, also referred to as TMV U2 strain)were propagated on Xanthi nn tobacco plants. Sunnhemp mosaic virus wasgrown in Phaseolus vulgaris. Virus was purified, and viral RNA wasprepared according to Bruening, G., et al. Virology, 71: 498-571, 1976.

To determine to what extent accumulation of MPΔ3-5 had an effect onviral infection and spread, seedlings from transgenic plant line3A5-SX-11 were inoculated with TMV (0.25 μg ml⁻¹) and analyzed fordevelopment of systemic symptoms in the upper leaves (FIG. 3). By thefifth day post-inoculation (DPI) plants that did not accumulate theMPΔ3-5 showed clear systemic symptoms of infection (FIG. 3a). By 7 DPIall or nearly all of the control plants displayed pronounced systemicsymptoms (FIG. 3a). In contrast, by 9 DPI no systemic symptoms wereproduced on the MPΔ3-5(+) plants (FIG. 3a). At 10 DPI 10% of theMPΔ3-5(+) plants exhibited symptoms, and by 13 DPI half of the MPΔ3-5(+)plants showed systemic symptoms (FIG. 3a). When the TMV inoculum wasraised fourfold (to 1.0 μg ml⁻¹), the MPΔ3-5(+) plants developedsystemic symptoms by 9 DPI, and by 13 DPI nearly all of the MPΔ3-5(+)plants exhibited symptoms due to TMV infection (FIG. 3b). However, thesymptoms that eventually developed on MPΔ3-5(+) plants upon TMVinfection were less severe than those produced by the control plants.Inoculating the plants with TMV-RNA resulted in essentially the sameresults, i.e. a pronounced delay of systemic symptom development onMPΔ3-5(+) plants compared with control plants (FIG. 3c). MPΔ3-5(+)plants inoculated with a related tobamovirus, tobacco mild green mosaicvirus (TMGMV) also showed a delay of systemic symptom development (FIG.3d).

To quantify virus accumulation in the upper systemically infectedleaves, the relative amount of TMV coat protein (CP) was measured atdifferent times after inoculation in leaf samples taken from the thirdleaf above the inoculated leaf (Table 5). While TMV was detected in theupper leaves of control plants at 5 DPI, no virus was detected inMPΔ3-5(+) plants at that day. At 9 DPI TMV accumulation was detected inthe MPΔ3-5(+) plants. However, the amount of virus detected in MPΔ3-5(+)plants was threefold lower than that in control plants (Table 5).

                  TABLE 5    ______________________________________    Relative accumulation of TMV in systemic leaves of infected    plants               Relative levels of               CP (c.p.m. 10 μg protein.sup.-1)    Line         5 DPI     9 DPI    ______________________________________    3A5+         0          471 (320)    3A5-         106 (48)  1388 (155)    WT           226 (19)  1399 (225)    ______________________________________     Xanthi nn plants were inoculated with TMV (1.0 μg/ml). The values give     are c.p.m. of .sup.125 Ilabeled secondary antibody bound to immunoblots     and represent the relative amount of TMP CP per 10 μg of protein     extracted from fresh leaf tissue as described in Experimental procedures.     Each value represents the average of 10 different samples. Values in     parentheses represent the standard error.     3A5+, transgenic plants expressing MPΔ35 (line 3A5SX-11);     3A5-, nonexpressing transgenic plants (segregants of line 3A5SX-11);     WT, wildtype Xanthi nn plants.

f) How to Determine that Resistance by Transgenic Plants ExpressingDysfunctional Viral Movement Protein is Due to the Reduction of VirusSpread and not Reduction of Virus Infection or Replication

To confirm that MPΔ3-5 reduced virus spread per se rather than virusinfection or replication the following experiment was performed. ATMV-based CDNA clone containing the gene encoding the reporter enzymeβ-glucuronidase (GUS) (Jefferson et al., Proc Natl Acad Sci USA, 88:2702-2706, 1986) in place of the MP gene was used to determine if, uponinoculation with TMV, MPΔ3-5(+) plants produced the same number ofinitial infection sites as non-transgenic plants.

Leaf disks (32 mm in diameter) taken from inoculated leaves wereimmersed in 1 ml GUS assay buffer (50 mM Na-phosphate buffer, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1% sucrose, 0.5 mM potassiumferrocyanide/ferricyanide, 10 mM β-mercaptoethanol) containing 0.5 mgml⁻¹ X-Gluc. The samples were vacuum infiltrated, and incubatedovernight at 37° C. The number of blue cells or areas were determined bymicroscopic examination.

The modified TMV containing the GUS gene (TMVΔM-GUS) was constructed bydeleting most of the MP gene sequence (nt 4923-5402) from the viral cDNAclone and there inserting the GUS coding sequence. Upon inoculation oftobacco plants with an in vitro transcript of TMVΔM-GUS, the transcriptretains the ability to replicate and produce virions, and in additioncauses accumulation of the GUS enzyme. The accumulation of GUS activitynecessarily reflects virus replication and the production of subgenomicRNAs, since translation of internal cistrons of TMV is known not tooccur (Beachy and Zaitlin, Virology, 81: 160-169, 1977). However,TMVΔM-GUS does not move from cell to cell since it lacks most of theviral MP gene sequence. Cells in which the enzyme accumulates produce ablue stain following incubation with the GUS substrate X-Gluc. Whentranscripts of TMVΔM-GUS were inoculated to MP(+) plants, infection andthe accumulation of GUS spread from cell to cell as expected due tocomplementation by the transgene. In contrast, when MPΔ3-5(+) plants ornon-transgenic plants were inoculated with TMVΔM-GUS only single cellsshowed GUS activity, indicating initial sites of viral replication ofblue cells were found on leaves of MPΔ3-5(+) and non-transgenic plantsinoculated with TMVΔM-GUS. This suggests that resistance in MPΔ3-5(+)plants was not due to inhibition of replication per se.

g) Analysis of Resistance to Viral Spread in Transgenic Local LesionHosts

A transgenic local lesion host which accumulates MPΔ3-5, plant line3A5-NN-7B, was also tested by inoculating R₁ seedlings with TMV,TMV-RNA, TMGMV and with a distantly related tobamovirus, SHMV. In allcases, MPΔ3-5(+) plants developed 1/4 to 1/7 the number of necroticlocal lesions compared with the control non-transgenic plants (Table 5).Despite the low amino acid sequence homology between the SHMV MP and theTMV MP (less than 30%), the number of lesions caused by SHMV was reducedas a result of the expression of MPΔ3-5. Moreover, the lesions thatdeveloped on MPΔ3-5(+) plants were significantly smaller in diameter (byabout 50%) than the lesions that developed on the control plants (FIG.4). Two of the R₁ seedling progeny of the 3A5-NN-7B plant line that didnot accumulate MPΔ3-5 developed the same number of lesions, and thelesions were of the same size, as those that developed on controlplants.

In all experiments there was a clear correlation between accumulation ofthe MPΔ3-5 protein and symptom inhibition, i.e. numbers and size ofnecrotic local lesions or a delay in systemic symptom development.

                                      TABLE 6    __________________________________________________________________________    Effect Of MPΔ3-5 on number of necrotic local lesion    Percentage AA.sup.b                   conc.                        Plant line  % of    Innoculum          identity (μg ml.sup.-1)                        3A5-NN-7B                              Xanthi NN                                    control    __________________________________________________________________________    TMV   100      0.25 10 (7)                              74 (40)                                    13.5    TMV   100      1.0  51 (42)                              190 (54)                                    26.8    TMV-RNA          100      1.3  36 (21)                              77 (30)                                    46.7    TMGMV  57      2.0   8 (7)                              40 (19)                                    20    SHMV.sup.a           29      10.0 69 (24)                              165 (49)                                    41.8    __________________________________________________________________________     The values given are number of necrotic local lesions per inoculated leaf     counted 4896 HPI of plants with virus or viral RNA. Each sample was     inoculated to 15 leaves.     Values in parentheses represent the standard error.     Conc. , concentration;     AA, amino acids.     .sup.a Lesions induced by SHMV were counted 8 DPI.     .sup.b The amino acids sequence comparison was done by allowing small     gaps.

Example 2 Determination of Multi-Virus Resistance in Transgenic TobaccoPlants that Express Dysfunctional Viral Movement Protein

This example demonstrates that tobacco plants expressing the TMVdysfunctional protein, MPΔ3-5, have the ability to restricted viralmovement and delay of disease symptoms when inoculated with otherrelated virus of the tobamovirus group as well as viruses from othergroups/families. Plants expressing MPΔ3-5 retard disease symptomdevelopment and reduce the accumulation of additional tobamoviruses andtwo viruses from the MP family one, tobacco rattle tobravirus (TRV) andpeanut chlorotic streak caulimovirus (PClSV), and a member from the MPfamily two, alfalfa mosaic ilarvirus (AlMV).

a) Plants

Transgenic Nicotiana tabacum cv. Xanthi nn lines 274 and 277 and N.tabacum cv. Xanthi NN line 2005 that express a gene encoding the TNV MP(MP+) were described by Deom, C. M., et al., 1987, supra. Transgenic N.tabacum cv. Xanthi NN line 3A5-SX-11 and N. tabacum cv. Xanthi nn lines3A5-NN-7B and 3A6-NN-2 expressing the gene encoding a MPΔ3-5 aredescribed above. Nicotiana tabacum cv. Xanthi nn line 3A7-SX-8, atransgenic plant line that does not express the MP or MPΔ3-5 genes(MP-), and nontransformed N. tabacum cv. Xanthi NN were used as negativecontrols. All plants were grown in a greenhouse (University ofCalifornia, Riverside) or in controlled environmental chambers asdescribed in Example 1.

b) Virus Propagation and Inoculation

Using methods commonly practiced in the art, tobamovirus TMV strain U1,tobamovirus Ob, which systemically infects N. tabacum Xanthi NN(Padgett, H. S. & Beachy, R. N., The Plant Cell 5, 577-586, 1993), Cg, acruciferous tobamovirus (kindly provided by M. Ishikawa and T. Ohno,Hokkaido University, Japan), tomato mosaic tobamovirus (ToMV), tobaccorattle tobravirus (kindly provided by D. J. Robinson, S.C.R.I.,Invergowrie, Scotland), and alfalfa mosaic ilarvirus were maintained inappropriate Nicotiana spp. Peanut chlorotic streak caulimovirus,generously provided by R. Richins, University of California, Riverside)was maintained in N. tabacum in a growth chamber at 32° C. Viruses wereidentified by inoculation to diagnostic host plants and by reactions tovirus specific antibodies. Source plant leaves were triturated, andextracts were diluted in inoculation buffer (20 mM phosphate buffer, pH7.0, 1 mM EDTA). Inoculum was applied to a single carborundum-dustedleaf on each of eight plants of lines 274, 277, 3A5-SX-11 and 3A7-SX-8or 2005, 3A5-NN-7B, 3A6-NN-2 and NN plants. Extracts were alsoinoculated onto Chenopodium amaranticolor to quantitate the infectivityof each inoculum. The average number of lesions on C. amaranticolorrepresented a relative inoculum potential (IP) for each of the virusestested. Only those plants inoculated with PCLSV were grown in a growthchamber at 32° C.

c) Symptom Development

Each leaf of each plant, including the inoculated leaf, was observed fordisease symptom appearance. The maximum score was 5, indicating that theplant had at least 5 leaves with disease symptoms; a score of zeroidentified a plant devoid of symptoms. A score of 1 reflected theappearance of symptoms on inoculated leaves. The intensity of symptomswas not scored. The average number of tobacco leaves that developedvisible symptoms on a plant was plotted against time (days afterinoculation or DAI). Analysis of variance of symptoms on the transgenicplants was compared to symptoms on negative controls. Differences at the95% level using the Fischer PLSD test were considered to be significant.

d) Quantitation of Virus

On the first day that systemic symptoms appeared on the control plantsinoculated with TMV, PClSV or AlMV, a 1 cm diameter disc was taken fromthe uppermost leaf (>5 cm in length) on all plants and discs from eachtreatment were pooled. The same leaf was sampled in subsequent days. Thepooled samples were triturated in 35 mM KH₂ PO₄ pH 7.5, 10 mM2-mercaptoethanol, 400 mM NaCl, and the total soluble protein in theextracts was determined using bicinchoninic acid protein assay reagent(Pierce, Rockford, Ill.). An equivalent amount of protein and differentamounts of the appropriate purified virus were separated by SDS-PAGE(Laemmli, U.K. Nature (London) 277, 680-685, 1977) in 12.5% gels andtransferred to nitrocellulose (Towbin, H., et al, Proc Natl Acad Sci USA76, 4350-4354, 1979). The membrane was treated with the appropriateviral coat protein rabbit antibody, followed by an alkaline phosphataseconjugated antirabbit antibody and developed with the ProtoBlot® II APSystem purchased from Promega Corp., supra. The amount of coat proteindetected on a Sci-Scan 5000 densitometer (United States Biochemical) wasinterpolated from a standard curve. TRV was quantitated by the followingbiological assay. The uppermost >5 cm leaf was sampled from plantinoculated with TRV after symptoms appeared on the control plants. Eachsample was ground in inoculation buffer to a standard dilution andinoculum was applied to leaves of C. amaranticolor. The leaves wereobserved several days later for the presence or absence of TRV-inducedlesions.

e) Plants Challenged with Tobamoviruses

As shown in Example 1, plant lines that accumulate MPΔ3-5 restricted thespread of TMV and two other tobamoviruses. To determine whether therewas similar resistance to these and other tobamoviruses under greenhouseconditions eight plants of lines 3A7-SX-8 (MP-), 274 (MP+), and3A5-SX-11 (MPΔ3-5+), all having the genotype nn, were inoculated withTMV and monitored for systemic disease symptoms over a 10 day period.The TMV inoculum produced an average of 50 lesions per leaf on C.amaranticolor and corresponded with the IP (inoculum potential) shown inExample 1 achieved with 0.25 ug/ml of TMV. Under greenhouse conditionssymptoms were delayed by 3-4 days on the 3A5-SX-11 line that accumulatedthe TMV MPΔ3-5 (Table 7). Immunoblot assays revealed that less virusaccumulated in the leaves above the inoculated leaves of plant line3A5-SX-11 than in either plant line 274 (which accumulates a functionalMP) or plant lines that do not accumulate a MP or a MPΔ3-5 (plant line3A7-SX-8). This data was similar to that in Example 1. Plant lines3A5-NN-7B (MPΔ3-5+) and 3A6-NN-2 (MPΔ3-5+) delayed systemic symptomdevelopment of tobamovirus Ob by 3 days as compared to negative controls(Table 7 and FIG. 5), while plants expressing a functional MP (2005)exacerbated symptom development of Ob. Inoculation of tobamovirus Cg toplant line 3A5-NN-7B (MPΔ3-5+) reduced the numbers of local lesions by78% at low IP and 56% at high IP (Table 7). Plant line 3A5-SX-11expressing the MPΔ3-5 delayed systemic symptom development of ToMV by 3days compared with control plants (Table 7).

                  TABLE 7    ______________________________________    Resistance in tobacco plants to tobamoviruses due to the DMP.    VIRUS  μg/ml  PLANT LINE  SYMPTOM*    ______________________________________    TMV-U1 0.25      3A5-SX-11   3 day delay of SS           0.25      3A5-SX-11   14 day delay of SS†           0.50      3A5-NN-7B   83% reduction in #LL           1.00      3A5-NN-7B   75% reduction in #LL†           0.50      3A6-NN-2    85% reduction in #LL           1.00      3A6-NN-2    91% reduction in #LL†    TMGMV  2.00      3A5-SX-11   3 day delay of SS           2.00      3A5-SX-11   14 day delay of SS†           2.00      3A5-NN-7B   80% reduction in #LL†    SHMV   10.00     3A5-NN-7B   58% reduction in #LL†    Ob     0.25      3A5-NN-7B   3 day delay of SS           0.25      3A6-NN-2    3 day delay of SS    Cg     1.00      3A5-NN-7B   78% reduction in #LL           2.00      3A5-NN-7B   56% reduction in #LL    ToMV   0.50      3A5-SX-11   3 day delay of SS    ______________________________________     *Data presented is relative to symptoms on the appropriate negative     controls. Plant lines designated SX are systemic hosts for all of these     tobamoviruses. Tobamovirus Ob does not cause necrotic local lesions on NN     plant lines, but all others do.     SS = systemic symptoms;     #LL = number of local lesions.     †Data frorn Lapodit, M., et al., The Plant Journal, 4:959-970,     1993, obtained under controlled artificial conditions. All other data     obtained under greenhouse conditions.

f) Plants Challenged with TRV

To assess resistance to TRV, virus was inoculated to Xanthi NN (MP-) andplant lines 2005 (MP+), 3A5-NN-7B (MPΔ3-5+) and 3A6-NN-2 (MPΔ3-5+). Whenplants were inoculated with an inoculum that produced an average of 260lesions per leaf on C. amaranticolor the inoculated leaves of alltobacco lines produced characteristic local necrotic reactions of equalseverity that appeared at the same time on each line. The control plantline (MP-) developed systemic symptoms by 7 DAI, but there were nosystemic symptoms on plant lines 3A5-NN-7B (MPΔ3-5+) or 3A6-NN-2(MPΔ3-5+) by 20 DAI (FIG. 6). In a second experiment in which the TRV IPwas sufficient to cause an average of 38 lesions on C. amaranticolor,leaves above the inoculated leaf were sampled from tobacco plants at 11DAI and extracts were inoculated to C. amaranticolor. Only extracts fromthe control plants induced large numbers of lesions indicative for TRVon C. amaranticolor. No virus was recovered from 8/8 of the 3A5-NN-7Bplants or 5/8 of the 3A6-NN-2 plants. Virus was recovered from 3/8 ofthe 3A6-NN-2 plants but lesion numbers were low (Table 8).

                  TABLE 8    ______________________________________    Systemic infection of plants inoculated with TRV.*                  # PLANTS SYSTEMICALLY    PLANT LINE    INFECTED/TOTAL PLANTS    ______________________________________    NN (MP-)      .sup.  8/8 †    2005 (MP+)    8/8    3A5-NN-7B (MPΔ3-5)                  6/8    3A6-NN-2 (MPΔ3-5)                  .sup.  3/8 ‡    ______________________________________     *At 11 DAI, upper leaves from plants inoculated with TRV were homogenized     in buffer and clarified extracts were inoculated to C. amaranticolor,     which were subsequentiy scored for the presence of lesions.     †Two tobacco plants that showed TRV symptoms yielded negative     bioassays     ‡One of the tobacco plants that yielded a positive bioassay wa     negative for TRV symptoms until 20 DAI. The two other plants showed low     virus accumulation.

g) Plants Challenged with PClSV

Plant lines 3A5-SX-11 (MPΔ3-5+), 274 (MP+), and 3A7-SX-8 (MP-) wereinoculated with leaf extracts containing the caulimovirus Pclsv and theplants were grown in a growth chamber at 32° C. Immunoblot analysis ofsamples collected from the third, sixth and ninth leaves above theinoculated leaf at 30 DAI did not detect virus in the MPΔ3-5(+) plants(Table 9). There was between 3 and 6 times as much virus in plantsexpressing a functional MP as compared to non-transgenic plants,suggesting that the MP exacerbated the spread or accumulation of PClSVin these plants. Symptoms were difficult to evaluate because of thechlorosis caused by the elevated temperatures.

                  TABLE 9    ______________________________________    Accumulation of Pclsv in upper tissues of transgenic plants.*                LOCATION OF LEAF ABOVE                INOCULATED LEAF    PLANT LINE    +3         +6       +9    ______________________________________    3A7-SX-8 (MP-)                  0          100      100    274 (MP+)     180        330      570    3A5-SX-11 (MPΔ3-5)                  0           0        0    ______________________________________     *Values are % of Pclsv extracted from the upper tissues of plants at DAI     30 per μg of tobacco plant protein as compared with virus in 3A7SX-8.     Virus was quantitated by immunoblot analysis.

h) Plants Challenged with AlMV

Studies with AlMV were performed on Xanthi nn and NN plant lines withinocula that produced high (>200) or low numbers of lesions on C.amaranticolor (Golemboski, D. B., et al., Proc Natl Acad Sci USA 87:6311-6315, 1990). In all studies the inoculated leaves of all tobaccolines developed the same number and type of local necrotic reactions atthe same time. At the high level of inoculum non-transgenic nn plantsand plants with MP (line 274) developed systemic symptoms within 4-6days. Plant line 3A5-SX-11 (MPΔ3-5+) developed symptoms 2 to 3 dayslater and eventually all plants developed symptoms. In a subsequentstudy both nn and NN plant lines were challenged with AlMV at a lowlevel of inoculum. All plant lines with the MPΔ3-5 gene developedsymptoms 5 to 10 days later than nontransgenic plants or plants with MP(FIGS. 7a and 7b). Immunoblot analysis of virus in extracts of leavesdemonstrated that there was little or no detectable AlMV in the tissuesabove the inoculated leaves of the plant lines 3A6-NN-2 or 3A5-SX-11that express MPΔ3-5 (Table 10); plant line 3A5-NN-7B delayed theaccumulation of virus in this study. These studies demonstrated that theMPΔ3-5 had little effect on the infection of inoculated leaves, but theMPΔ3-5 did retard or block the spread of AlMV to upper leaves.

                  TABLE 10    ______________________________________    Accumulation of AlMV in upper tissues of transgenic plants.*                DAYS AFTER INOCULATION    PLANT LINE    7          8        9    ______________________________________    NN (MP-)      37         46       56    2005 (MP+)    125        159      77    3A5-NN-7B (MPΔ3-5)                   3         28       54    3A6-NN-2 (MPΔ3-5)                   0          0        0    3A7-SX-8 (MP-)                  34         70       53    274 (MP+)     28         51       42    3A5-SX-11 (MPΔ3-5)                   0          0        0    ______________________________________     *Numbers are ng of AlMV extracted from the pooled samples of the upper     tissues of plants from FIGS. 3a and 3b per μg of total plant protein a     determined by immunoblot analysis.

Example 3 Transient Assays for the Identification of Mutant ViralMovement Proteins that Block Virus Spread

Two types of transient assays can be utilized for the identification oftransgenically expressed mutant viral movement proteins capable ofinterfering with the spread of virus infection. Both assays involveanalysis for the development of disease symptoms that result aftersimultaneous expression of MP and the candidate mutated viral movementprotein. In one assay symptoms are analyzed in plants that have beensimultaneously inoculated with equal amounts of two infectious clones,one clone containing MP and the other containing the candidate mutatedviral movement protein. In the second assay, symptoms are analyzed inplants inoculated with an infectious clone containing a cassette thatcauses the production of equal numbers of molecules of the two proteins,MP and candidate mutated viral movement protein. In both assays, if themutated movement protein is capable of interfering with the wtmp andblocks the intercellular movement of virus, the spread of diseasesymptoms from the initial site of inoculation will be stopped ordelayed.

a) Transient Assay Incorporating Separate Infectious Clones

This assay requires the preparation of an infectious clone of the virusof interest. To prepare the infectious clone one prepares a CDNA copy ofthe viral genome. To make the clone infectious, an appropriatetranscriptional, e.g., T7 promoter, is inserted into the clone at apoint that under the appropriate in vitro conditions there is synthesisof RNA that can cause infection when inoculated to an appropriate host.An alternative approach is to place the viral CDNA downstream of atranscriptional promoter that is active in plant cells causing virusinfection when transfected into plant cells.

Two infectious clones are constructed; the first containing the MP andthe second containing the mutant MP. Mutant and wild type infectiousRNAs are coinoculated in ratios that lead to a high probability thatmost or all cells are co-infected by both. Co-infection is commonamongst viruses that are co-inoculated but it should be noted thatattempts to sequentially infect plants can be unsuccessful. If themutant MP acts in a dominant manner and prevents the MP fromfunctioning, the infection will be limited, and may either result insites of infection that are restricted in size or the infection will berestricted of slowed in systemic spread.

b) Transient Assay Incorporating Expression Cassette that Produces EqualAmounts of MP and Mutant MP

This assay relies upon a genetically engineered TMV that contains anexpression cassette that causes the production of equal numbers of twoproteins from a single promoter. The preparation of the cassette isdescribed by Marcos and Beachy, Plant Molecular Biology 24: 495:5031994. In brief, the cassette (PPRO1) includes the tobacco etch virus(TEV) nuclear inclusion (NIa) proteinase coding region flanked on eachside by its corresponding heptapeptide cleavage sequence along withcloning sites for in frame insertion of two different open readingframes. The cassette allows the synthesis, under the control of a singletranscriptional promoter, of two proteins in equimolar amounts as partof a polyprotein which is cleaved into individual mature products by theTEV protease.

In the current assay the cassette will be constructed to encode the wtMPalong with the mutant movement protein. Using commonly practiced methodsof genetic engineering, the gene cassette is introduced into the genomeof TMV (or other viruses) in place of the gene encoding the wild typeMP. Using the methods described in examples 1 and 2, tobacco plants areinoculated with this engineered TMV. For a control, wild type TMV isused as the inoculum. The plants are then analyzed for the developmentof disease symptoms. If the mutant MP interferes with the MP, infectionswill be confined to small foci and will either not spread locally orwill do so at a slower rate when compared with the controls. When thisoccurs the mutant MP genes are candidates for use as virus resistantgenes and can be further tested in transgenic tobacco plants.

It is apparent to those skilled in the art that a similiar strategycould also be taken to incorporate the polyprotein cleavage cassetteinto the cloned cDNA for viruses other than TMV.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A method of producing a viral resistant plantsaid method comprising the step of transforming the plant with a geneencoding a viral movement protein derived from tobacco mosaic virus,wherein said viral movement protein is mutated between amino acids 2 and8 in the amino terminus, and wherein said protein is able to bind theplasma membrane and cell walls of the plant and inhibit theintercellular and/or systemic movement of a virus selected from thegroup consisting of Ilar viruses, bromovirus, caulimovirus, hordeivirus,luteovirus, tobamovirus and tospovirus.
 2. The method of claim 1 whereinsaid inhibited virus is a Tobamovirus.
 3. The method of claim 1 whereinthe mutation is a deletion of amino acids 3-5.
 4. The method of claim 1wherein said inhibited virus is a Tobamovirus.
 5. The method of claim 4wherein the mutation is a deletion of amino acids 3-5.